PROJECT SUMMARY More potent drugs that seek to eradicate the more drug resistant forms of Methicillin-resistant Staphyloccocus aureus (MRSA), a devastating skin infection, only promotes further drug resistance. An alternate treatment is necessary, and antivirulence could be that treatment. By inhibiting antivirulence factors in the accessory gene regulator (agrA), the quorum sensing pathway is quenched. This suppresses aggression of the pathogen, which allows the host to clear the infection. ?-Hydroxyemodin has shown success inhibiting MRSA skin and soft tissue infections in an in vivo mouse model through this pathway by binding to agrA, the response regulator. Because further testing is necessary before a drug can reach hospitals, a greater supply as well as alternate analogues is necessary. This project seeks to not only increase the supply of ?-hydroxyemodin from Penicillium restrictum, a known producer of the compound, but also examine the other faculties of the microorganism that may produce analogues that show equal or greater activity than ?-hydroxyemodin. By changing the culture conditions and retaining the same fungus, the biosynthetic pathway for polyhydroxyanthraquinones will remain but the types of polyhydroxyanthraquinones produced will change. Profiles of each condition will be rapidly accessed with droplet-liquid microjunction-surface sampling probe (droplet-LMJ-SSP). The profiles can be compared not only to determine optimal growing conditions for the production of the key active compound but also significant differences in metabolite profile to merit further investigation. These compounds will be tested for inhibition of MRSA and characterized via high resolution mass spectrometry and a suite of NMR techniques. This study combines the one strain-many compounds approach to stress and change the secondary metabolite production of the fungus, droplet-liquid microjunction- surface sampling probe to examine the secondary metabolites in situ, and chemometrics and mass defect filtering to characterize and analyze secondary metabolite profiles.